The NEDD8 system is essential for cell cycle progression and morphogenetic pathway in mice.

Tateishi K, Omata M, Tanaka K, Chiba T - J. Cell Biol. (2001)

Bottom Line:
Uba3(-/-) mice died in utero at the periimplantation stage.Mutant embryos showed selective apoptosis of the inner cell mass but not of trophoblastic cells.However, the mutant trophoblastic cells could not enter the S phase of the endoreduplication cycle.

ABSTRACTNEDD8/Rub1 is a ubiquitin (Ub)-like molecule that covalently ligates to target proteins through an enzymatic cascade analogous to ubiquitylation. This modifier is known to target all cullin (Cul) family proteins. The latter are essential components of Skp1/Cul-1/F-box protein (SCF)-like Ub ligase complexes, which play critical roles in Ub-mediated proteolysis. To determine the role of the NEDD8 system in mammals, we generated mice deficient in Uba3 gene that encodes a catalytic subunit of NEDD8-activating enzyme. Uba3(-/-) mice died in utero at the periimplantation stage. Mutant embryos showed selective apoptosis of the inner cell mass but not of trophoblastic cells. However, the mutant trophoblastic cells could not enter the S phase of the endoreduplication cycle. This cell cycle arrest was accompanied with aberrant expression of cyclin E and p57(Kip2). These results suggested that the NEDD8 system is essential for both mitotic and the endoreduplicative cell cycle progression. beta-Catenin, a mediator of the Wnt/wingless signaling pathway, which degrades continuously in the cytoplasm through SCF Ub ligase, was also accumulated in the Uba3(-/-) cytoplasm and nucleus. Thus, the NEDD8 system is essential for the regulation of protein degradation pathways involved in cell cycle progression and morphogenesis, possibly through the function of the Cul family proteins.

fig6: Impaired cell cycle regulation in Uba3−/− embryos. Cultured blastocysts (A–F) or E5.75 embryos in utero (G–J) were treated with BrdU. BrdU incorporation was detected by immunohistochemistry. The confocal images of BrdU staining focused on the trophoblastic cells (A and B) or the ICM (C and D) were merged with the differential interference contrast image. In trophoblastic cells, Uba3+ (A) but not Uba3−/−(B) could enter S phase. In the ICM, both Uba3+ (C) and Uba3−/− (D) could enter this phase. Nuclei were counterstained with Hoechst 33342. Uba3−/−trophoblastic cells had smaller nuclei (F), whereas Uba3+ cells contained large nuclei (E). In in vivo experiments, BrdU-positive cells were evident in most part of Uba3+ embryos (G), whereas few cells were positive in Uba3−/−embryos (H). The serial sections were counterstained with H&E (I and J). Bars, 100 μm.

Mentions:
To identify cell cycle defects in Uba3-deficient cells, we added BrdU to the in vitro culture medium and tested whether Uba3−/− cells can enter the S phase. In trophoblastic cells, Uba3+ (Fig. 6 A) but not Uba3−/− (Fig. 6 B) could enter S phase. In the ICM, both Uba3+ (Fig. 6 C) and Uba3−/− (Fig. 6 D) could enter this phase. DNA staining revealed that most Uba3−/− trophoblastic cells contained small nuclei (Fig. 6 F), whereas Uba3+ cells contained large nuclei (Fig. 6 E), a morphological hallmark of endoreduplication (Varmuza et al., 1988). To assure the S phase entry is indeed impaired in vivo, we injected BrdU in 5.75 d postcoitum pregnant female and analyzed BrdU incorporation in the embryos. In wild-type embryos, BrdU-positive cells were observed in most parts of the embryonic ectoderm and extraembryonic tissue (Fig. 6, G and I). In contrast, mutant embryos showed few BrdU-positive cells (Fig. 6, H and J). Since the endoreduplication cycle requires fluctuation of CDKs activity (Traas et al., 1998), we next investigated whether any cell cycle regulators are dysregulated in trophoblasts. Cyclin E, a member of G1 cyclins, has been reported to be expressed periodically in trophoblastic cells, and this periodicity is essential for endoreduplication (MacAuley et al., 1998; Weiss et al., 1998). In Uba3+ cells, cyclin E expression was evident in a few trophoblasts consistent with the cell cycle–regulated expression pattern of cyclin E (Fig. 7 A), whereas overexpression of cyclin E was observed in most of Uba3−/− trophoblasts (Fig. 7 B). A similar accumulation of cyclin E was also observed in the ICM of Uba3−/− embryos (Fig. 7 D) compared with Uba3+ embryos (Fig. 7 C). Immunostaining of other cyclins revealed that cyclin D3, another member of G1 cyclins, was highly expressed compared with wild-type littermates. Since overexpression of cyclin E normally assists in overriding the G1-S checkpoint rather than G1-S arrest (Lukas et al., 1997), we next tested the expression of CKIs that negatively regulate CDK activity. Among the member of CKIs, the expression of p57Kip2 is known to be induced upon differentiation of trophoblasts, and its level fluctuates during the endoreduplication cycle (Hattori et al., 2000). Indeed, p57Kip2 was expressed abundantly in most Uba3−/− trophoblasts (Fig. 7 F) but only in part of Uba3+ trophoblasts (Fig. 7 E). On the other hand, p57Kip2 was expressed in a few cells in the ICM of both Uba3+ and Uba3−/− embryos (Fig. 7, G and H). These findings are consistent with those of a recent study, indicating that periodic expression of p57Kip2 is required for the next S phase entry from the G2-like phase of the endoreduplication cycle (Hattori et al., 2000). No significant accumulation of other members of CKI, p21Cip1, and p27Kip1 was noted in trophoblasts or ICM (unpublished data). Thus, S phase entry of Uba3−/− trophoblasts is blocked in part due to accumulation of p57Kip2, or alternatively trophoblasts are arrested before the point that p57Kip2 degrades. Intriguingly, cyclin E accumulated in Cul-3−/− trophoblasts as well, which failed to endoreduplicate (Singer et al., 1999), suggesting that Cul-3 function might be impaired in Uba3−/− mice.

fig6: Impaired cell cycle regulation in Uba3−/− embryos. Cultured blastocysts (A–F) or E5.75 embryos in utero (G–J) were treated with BrdU. BrdU incorporation was detected by immunohistochemistry. The confocal images of BrdU staining focused on the trophoblastic cells (A and B) or the ICM (C and D) were merged with the differential interference contrast image. In trophoblastic cells, Uba3+ (A) but not Uba3−/−(B) could enter S phase. In the ICM, both Uba3+ (C) and Uba3−/− (D) could enter this phase. Nuclei were counterstained with Hoechst 33342. Uba3−/−trophoblastic cells had smaller nuclei (F), whereas Uba3+ cells contained large nuclei (E). In in vivo experiments, BrdU-positive cells were evident in most part of Uba3+ embryos (G), whereas few cells were positive in Uba3−/−embryos (H). The serial sections were counterstained with H&E (I and J). Bars, 100 μm.

Mentions:
To identify cell cycle defects in Uba3-deficient cells, we added BrdU to the in vitro culture medium and tested whether Uba3−/− cells can enter the S phase. In trophoblastic cells, Uba3+ (Fig. 6 A) but not Uba3−/− (Fig. 6 B) could enter S phase. In the ICM, both Uba3+ (Fig. 6 C) and Uba3−/− (Fig. 6 D) could enter this phase. DNA staining revealed that most Uba3−/− trophoblastic cells contained small nuclei (Fig. 6 F), whereas Uba3+ cells contained large nuclei (Fig. 6 E), a morphological hallmark of endoreduplication (Varmuza et al., 1988). To assure the S phase entry is indeed impaired in vivo, we injected BrdU in 5.75 d postcoitum pregnant female and analyzed BrdU incorporation in the embryos. In wild-type embryos, BrdU-positive cells were observed in most parts of the embryonic ectoderm and extraembryonic tissue (Fig. 6, G and I). In contrast, mutant embryos showed few BrdU-positive cells (Fig. 6, H and J). Since the endoreduplication cycle requires fluctuation of CDKs activity (Traas et al., 1998), we next investigated whether any cell cycle regulators are dysregulated in trophoblasts. Cyclin E, a member of G1 cyclins, has been reported to be expressed periodically in trophoblastic cells, and this periodicity is essential for endoreduplication (MacAuley et al., 1998; Weiss et al., 1998). In Uba3+ cells, cyclin E expression was evident in a few trophoblasts consistent with the cell cycle–regulated expression pattern of cyclin E (Fig. 7 A), whereas overexpression of cyclin E was observed in most of Uba3−/− trophoblasts (Fig. 7 B). A similar accumulation of cyclin E was also observed in the ICM of Uba3−/− embryos (Fig. 7 D) compared with Uba3+ embryos (Fig. 7 C). Immunostaining of other cyclins revealed that cyclin D3, another member of G1 cyclins, was highly expressed compared with wild-type littermates. Since overexpression of cyclin E normally assists in overriding the G1-S checkpoint rather than G1-S arrest (Lukas et al., 1997), we next tested the expression of CKIs that negatively regulate CDK activity. Among the member of CKIs, the expression of p57Kip2 is known to be induced upon differentiation of trophoblasts, and its level fluctuates during the endoreduplication cycle (Hattori et al., 2000). Indeed, p57Kip2 was expressed abundantly in most Uba3−/− trophoblasts (Fig. 7 F) but only in part of Uba3+ trophoblasts (Fig. 7 E). On the other hand, p57Kip2 was expressed in a few cells in the ICM of both Uba3+ and Uba3−/− embryos (Fig. 7, G and H). These findings are consistent with those of a recent study, indicating that periodic expression of p57Kip2 is required for the next S phase entry from the G2-like phase of the endoreduplication cycle (Hattori et al., 2000). No significant accumulation of other members of CKI, p21Cip1, and p27Kip1 was noted in trophoblasts or ICM (unpublished data). Thus, S phase entry of Uba3−/− trophoblasts is blocked in part due to accumulation of p57Kip2, or alternatively trophoblasts are arrested before the point that p57Kip2 degrades. Intriguingly, cyclin E accumulated in Cul-3−/− trophoblasts as well, which failed to endoreduplicate (Singer et al., 1999), suggesting that Cul-3 function might be impaired in Uba3−/− mice.

Bottom Line:
Uba3(-/-) mice died in utero at the periimplantation stage.Mutant embryos showed selective apoptosis of the inner cell mass but not of trophoblastic cells.However, the mutant trophoblastic cells could not enter the S phase of the endoreduplication cycle.

ABSTRACTNEDD8/Rub1 is a ubiquitin (Ub)-like molecule that covalently ligates to target proteins through an enzymatic cascade analogous to ubiquitylation. This modifier is known to target all cullin (Cul) family proteins. The latter are essential components of Skp1/Cul-1/F-box protein (SCF)-like Ub ligase complexes, which play critical roles in Ub-mediated proteolysis. To determine the role of the NEDD8 system in mammals, we generated mice deficient in Uba3 gene that encodes a catalytic subunit of NEDD8-activating enzyme. Uba3(-/-) mice died in utero at the periimplantation stage. Mutant embryos showed selective apoptosis of the inner cell mass but not of trophoblastic cells. However, the mutant trophoblastic cells could not enter the S phase of the endoreduplication cycle. This cell cycle arrest was accompanied with aberrant expression of cyclin E and p57(Kip2). These results suggested that the NEDD8 system is essential for both mitotic and the endoreduplicative cell cycle progression. beta-Catenin, a mediator of the Wnt/wingless signaling pathway, which degrades continuously in the cytoplasm through SCF Ub ligase, was also accumulated in the Uba3(-/-) cytoplasm and nucleus. Thus, the NEDD8 system is essential for the regulation of protein degradation pathways involved in cell cycle progression and morphogenesis, possibly through the function of the Cul family proteins.